KR20040025906A - Composition for the prevention of osteoporosis comprising a combination of isoflavones and polyunsaturaed fatty acids - Google Patents

Abstract

The present invention relates to (a) one or more isoflavones and / or isoflavone glycosides, preferably genistein and / or genistin; (b) one or more polyunsaturated fatty acids; (c) optionally, vitamin D and / or one or more derivatives thereof and / or vitamin K and / or one or more derivatives thereof; And (d) optionally, an adjuvant and an excipient in the required amount, preferably in the range of 0.1 to 20% by weight, based on the total weight of the composition. The present invention also relates to dietary compositions and herbal forms prepared from the compositions, and to the use of such compositions for preventing osteoporosis and stimulating bone formation in mammals.

Description

COMPOSITION FOR THE PREVENTION OF OSTEOPOROSIS COMPRISING A COMBINATION OF ISOFLAVONES AND POLYUNSATURAED FATTY ACIDS}

The bone consists mainly of collagen and calcium phosphate. Collagen is a protein that provides a soft skeleton, and calcium phosphate is a mineral that adds strength to the skeleton and hardens it. The mixture of collagen and calcium makes the bones strong and pliable to withstand pressure.

During life, the old bone is reabsorbed and new bone is formed. During childhood and teenage years, the new bone is formed more quickly than the old bone is removed. By about 25 years of age and reaching their thirties, bone resorption begins to slowly exceed bone formation resulting in bone loss, ultimately leading to osteoporosis.

Bone loss is more pronounced in women than in men. In women, bone loss accelerates for 10 years immediately after postmenopausal estrogen deficiency, but osteoporosis is age-related for both men and women and causes bone fractures and susceptibility by increasing bone destruction and susceptibility by reducing bone mass and microstructure deterioration of bone tissue. Is a systemic skeletal disease. The same menopausal condition as described above is found in female pets, such as dogs or cats, that cause bone loss due to estrogen deficiency after infertility surgery.

It has been found that compositions comprising isoflavones mixed with LC-polyunsaturated fatty acids (PUFA) have an effective additive and synergistic effect of preventing osteoporosis and stimulating bone formation. In addition, these effects are increased when the composition contains a predetermined amount of vitamin D and vitamin K. Surprisingly, ingestion of Genistein, PUFA and a predetermined amount of Vitamin K and Vitamin D completely prevents the loss of bone mineral density caused by estrogen deficiency in the OVX-rat model.

Isoflavones are 3-phenyl-benzo-γ-pyrone. Isoflavones commonly known in nature are substituted with hydroxy or methoxy. Preferred isoflavones within the scope of the present invention are genistein, which is the 4 ', 5,7-trihydroxy-isoflavone of formula I, wherein the genistein is, for example, soybean (e.g., Soya hispida ). Is known as its glycosidic form geninistin:

Polyunsaturated fatty acids are known per se (see EA Trautwein, Eur. J. Lipid Sci. Technol ., 103 , 45-55, 2001).

The present invention is intended to prevent osteoporosis in the form of concentrates, dietary compositions such as fortified foods, fortified feeds and beverages, or in the form of various herbal preparations such as tablets, granules filled in capsules, or effervescent preparations. It relates to a composition for.

The invention is defined in the claims. In particular, the present invention relates to (a) one or more isoflavones and / or isoflavone glycosides, preferably genistein and / or genistin; And (b) one or more polyunsaturated fatty acids, (c) vitamin D and / or one or more derivatives thereof and / or vitamin K and / or one or more derivatives thereof; And (d) auxiliaries and excipients in the range of 0.1 to 20% by weight, based on the required amount, preferably total weight of the composition.

The composition is obtained in the form of a concentrate, such as a simple powder mixture of ingredients, in the form of granules obtained by spray drying an aqueous slurry of ingredients or by extruding the mixture, compressing the powder into tablets by conventional tableting methods and machinery In the form of tablets, in the form of a paste filled in hard or soft gelatin capsules, or in the form of a foamable preparation. The present invention also relates to a method of preparing the composition.

The invention further relates to the use of said composition in the form of a concentrate for preparing a dietary composition or herbal form, wherein said dietary composition is preferably a fortified food, a fortified feed or a beverage, and the herbal form is preferably a tablet. , Granules, paste filled in hard or soft gelatin capsules, or foam preparations.

The invention also relates to a dietary composition or herbal preparation, wherein the dietary composition is preferably a fortified food, a fortified feed or a beverage, and the herbal preparation is preferably a paste filled in tablets, granules, hard or soft gelatin capsules, Or in the form of a foamable preparation, the dietary composition and the herbal preparation each comprising components (a), (b), (c) and (d) as defined above.

The invention also relates to the use of the composition for preventing osteoporosis and stimulating bone formation in mammals, such as but not limited to humans and pets (including, but not limited to cats and dogs).

The invention also relates to a method for preventing osteoporosis and stimulating bone formation in a mammal comprising administering to said mammal an effective amount of said composition as defined above.

Various isoflavones are known, for example dydzein, glycytein or genistein. Genistein is known, for example, as Genistin in its glycoside form in soybeans. According to the invention, the isoflavones themselves or isoflavone derivatives (eg glycosides) yielding isoflavones can be used as active ingredients [component (a)] in the composition.

Preferably, the composition comprises isoflavones and / or derivatives thereof at a concentration having a daily consumption in the range of 1 to 500 mg, preferably 5 to 100 mg.

Polyunsaturated fatty acids (PUFAs) are known per se. Preferred PUFAs are PUFAs having 16 to 24 carbon atoms, preferably 18 to 22 carbon atoms, more preferably 18, 20 or 22 carbon atoms, and having a plurality of unsaturated carbon-carbon double bonds.

Examples of such unsaturated fatty acids are known m-3 PUFAs. Linoleic acid, for example (cis, cis, cis-) 9,12,15-octadecationic acid, linolenic acid, for example (cis, cis, cis-) 6,9,12-octadecattric acid, Icosatetranic acid, icosapentaenoic acid and docosahexaenoic acid, for example (cis-) 5,8,11,14-icosatetranic acid, (cis-) 5,8,11,14,17 Preference is given to PUFAs having 18 carbon atoms, such as icosapentaenoic acid and / or (cis-) 4,7,10,13,16,19-docosahexaenoic acid.

Preferably the composition comprises PUFA at a concentration having a daily consumption of 50 to 8000 mg, preferably 500 to 2000 mg. Preferably, the composition comprises about 5 to 200 mg, preferably about 20 to 50 mg of polyunsaturated fatty acid [component (b)] for 1 mg of isoflavone or isoflavone derivative [component (a)].

The active metabolite of vitamin D (calcitriol) is known to increase intestinal absorption of calcium and is important for normal mineralization of the renal bone. The presence of vitamin D, preferably vitamin D ₃ , significantly improves the effect of the composition according to the invention. Composition each preferably includes vitamin D, more preferably D ₃ of a known concentration with respect to the vitamin D and vitamin D _3. The concentration should have vitamin D and vitamin D ₃ with daily consumptions ranging from 5 to 50 μg, respectively.

The classic role of vitamin K is as an anti-hemorrhagic factor. However, vitamin K is also a cofactor for this protein, which also interacts with selected proteins and plays a role in bone formation. The presence of vitamin K, preferably vitamin K ₁ , enhances the effect of the composition according to the invention. Preferably, the composition comprises vitamin K in a concentration known per se for vitamin K. The concentrations of vitamin K and vitamin K ₁ should each have a daily consumption in the range of 0.050 to 10 mg, preferably 0.1 to 1.0 mg.

As used herein, the term "vitamin D" refers to both vitamin D and vitamin D ₃ and derivatives thereof. The term "vitamin K" means both vitamin K and vitamin K ₁ and corresponding derivatives thereof, respectively.

Adjuvants may be optionally added. Suitable auxiliaries are, for example, starch, starch derivatives, cellulose, cellulose derivatives (eg hydroxypropylmethylcellulose [HPMC] and methylcellulose [MC]) and polyols. It is preferred not to add adjuvants to the concentrate.

The composition of the present invention can be prepared by any known method. The components can be mixed together simply by conventional methods. However, the concentrate is preferably prepared in powder form or granule form. Fluid bed granulation, high shear granulation, extrusion molding, spray drying or wet granulation are preferred.

When obtaining the composition of the present invention by spray drying, it is convenient to make all the components into an aqueous slurry. This slurry preferably has a solids content of about 10 to 70% by weight, more preferably about 25 to 50% by weight. The slurry is then spray dried by a method known per se. When obtaining the concentrate of the invention by fluid bed granulation, it is convenient to use a known fluid bed granulation device comprising a fluid bed drying device equipped with spraying means and operating in a manner known to those skilled in the art.

Concentrates can also be used to prepare dietary compositions, such as fortified foods, fortified feeds or beverages. These fortified foods, fortified feeds and beverages are known in the literature or are known to those skilled in the art.

The concentrate can also be compressed into tablets by conventional tableting methods and machinery. Optionally, the powder or granules may be formulated with conventional lubricants (e.g., metal stearates, stearic acid, vegetable hardening oils (Sterotex), glyceryl monostearate, glyceryl palmitostearate, talc, corn starch). , Polyethylene glycol, sodium benzoate, sodium acetate and sugar esters, but not limited thereto) and then compressed into tablets. In addition, the powder or granules may be mixed with the mixture of lubricants and then compressed into tablets.

Pastes, or foamable formulations, filled in hard or soft gelatin capsules are prepared by conventional methods, and these single units contain the same dosage as defined for tablets.

Experimental part

In the experimental section below, dual-energy X-ray absorption (DXA) was used to measure bone mineral density.

Osteocalcin, a biochemical marker for bone formation, is a bone-specific protein that is mainly secreted from osteoblasts, ie osteoblasts. Serum esteocalcin was measured as a marker for bone formation.

Deoxypyridinolin (DPD) cross-linking excretion provides a highly specific marker for bone resorption. DPD crosslinked excreta were measured as markers for bone resorption. This value should be corrected for urine concentration by creatinine measurements known in the art.

In the experimental setup, data were generated using an ovarian isolated rat osteoporosis (OVX) model. This model mimics the estrogen deficiency state found in postmenopausal women that cause osteoporosis or female pets after infertility surgery. The SHAM group with complete ovaries served as a control. Each test group consists of 10 to 12 animals. The feed used is free of isoflavones such as genistein. Test compounds of the various mixtures shown in Table 1 were administered as feed mixtures. In rats, administration of Genistein at 15 mg / kg body weight per day results in plasma Genistein levels similar to those found in Japanese people who consume traditional diets, ie 280 nM. The administration of 20 μg of vitamin K ₁ per kg of feed corresponds to a daily dose of about 1 μg per kg of body weight, which is a daily required calculation of vitamin K ₁ . The amount of vitamin D ₃ was 500 IU per kg of feed, corresponding to the daily dose of 25 IU per kg of body weight, the daily requirement calculation. In the case of reinforcement, PUFA (5% ROPUFA "30" n-3 Food Oil) was added to the feed corresponding to the% PUFA reinforcement in the feed as shown in Table 1. Bone mineral density, osteocalcin and deoxypyridinolin were measured 84 days after intervention.

Concentrations of Test Compounds in Experimental Group and Feed Military number SHAM / OVX Feed description Genistein (mg / kg body weight) N-3 PUFA in feed (%) Vitamin K ₁ (µg / kg Feed) Vitamin D ₃ (IU / kg Feed) One SHAM Vitamin K + D Supplement 2000 1500 2 OVX Vitamin K + D Supplement 2000 1500 3 OVX 20 500 4 OVX n-3 PUFA 5 20 500 5 OVX Genistein 15 20 500 6 OVX Vitamin K + D Supplements n-3 PUFA Genistein 15 5 2000 1500 SHAM: complete ovary model (control); OVX: uterine extractable rat osteoporosis model; Genistein: Roche 24-2076, Roche Vitamins AG, Basel, Switzerland; PUFA: Roche Vitamins, Basel, Switzerland AGRO's ROPUFA "30" n-3 food oil.

Measurement of Bone Mineral Density (BMD) in the Femur of OVX Rats

Compared to the SHAM operated animals of group 1, the ovarian extractable animals of groups 2 and 3 for large or small amounts of vitamin K and D diets showed an effective reduction in BMD as expected. Group 2 for large amounts of vitamin K and D feeds did not effectively preserve BMD better than animals in group 3 for small amounts of vitamin K and D feeds.

Animals in group 4 for small amounts of vitamin K and D feed supplemented with PUFA showed effective BMD retention compared to group 3 for small amounts of vitamin K and D feed. However, this group did not preserve effectively better than group 2 for large amounts of vitamins K and D without supplementing PUFA. At the same time, the BMD of group 4 was not significantly different from the BMD of SHAM operated animals. The same results as group 5 were obtained for small amounts of vitamin K and D feed supplemented with genistein. Group 6 for large amounts of vitamins K and D supplemented with PUFA and genistein in the mixture showed an effectively improved BMD compared to groups 2 and 3. No significant difference was found in BMD compared to Control 1. The results are shown in Table 2 below. Supplementation of n-3 PUFA or genistein improved BMD to control values, but did not effectively surpass the effects of vitamin D and vitamin K supplementation. Supplementation of vitamins K and D in a composition comprising n-3 PUFA and genistein restored maximally BMD that effectively exceeded the effects of vitamin D and K supplementation, even slightly exceeding BM in SHAM-operated control animals.

Bone Mineral Density of the Femur 84 Days After Intervention Military number SHAM / OVX Feed description Average (g / cm ² ) Standard Deviation Significant difference (*) One SHAM Vitamin K + D Supplement 0.143 0.006 a 2 OVX Vitamin K + D Supplement 0.135 0.012 bc 3 OVX 0.133 0.004 b 4 OVX n-3 PUFA 0.142 0.011 ac 5 OVX Genistein 0.143 0.013 ac 6 OVX Vitamin K + D Supplements n-3 PUFA Genistein 0.145 0.011 a (*) Different letters indicate significant differences between groups (p <0.05).

Serum Osteocalcin (Bone Formation Markers) Measurements in OVX Rats

Estrogen deficiency increased bone turnover causing high levels of serum osteocalcin. Compared to the control group, Groups 2 and 3 effectively increased osteocalcin levels, whereas the group's osteocalcin levels for small amounts of vitamin K and D feed were again effectively higher than the large amounts of vitamin D and K levels.

Supplementation of n-3 PUFA or genistein for groups 4 and 5 in addition to small amounts of vitamin K and D levels reduced osteocalcin levels to control values. There was no significant difference in the effect between n-3 PUFA and genistein. Group 6 on feeds of large amounts of vitamins K and D, including n-3 PUFA and genistein, showed the best effect on osteocalcin levels and was better than group 4. The results are shown in Table 3 below:

Serum concentration of serum osteocalcin 84 days after intervention Military number SHAM / OVX Feed description Mean serum concentration (ng / ml) Standard Deviation Significant difference (*) One SHAM Vitamin K + D Supplement 15.5 2.7 bd 2 OVX Vitamin K + D Supplement 28.4 4.8 c 3 OVX 39.6 4.9 a 4 OVX n-3 PUFA 17.8 2.9 d 5 OVX Genistein 14.6 3.8 bd 6 OVX Vitamin K + D Supplements n-3 PUFA Genistein 12.8 3.6 b (*) Different letters indicate significant differences between groups (p <0.05).

Deoxypyridinolin (bone resorption marker) in OVX rats

Estrogen deficiency significantly increased deoxypyridinolin (DPD) excretion that was effectively higher in the group for small amounts of vitamin K and D compared to the group for feeds with large amounts of vitamin K and D. The effect of n-3 PUFA supplementation was similar to that of large amounts of vitamin K and D feed. Genistein (Group 5), including feed with small amounts of vitamin K and D levels, returned the DPD feces to control values. In addition, group 6 was not effectively improved compared to group 5. The results are shown in Table 4 below, which shows that although Genistein is most effective at restoring control DPD excretion values among the tested compounds (Group 5), the addition of n-3 PUFAs to the feed (Group 4) resulted in large amounts of vitamin K and Indicates that the effects obtained by the D diet (group 2) were not surpassed:

Deoxypyridinolin feces 84 days after intervention Military number SHAM / OVX Feed description Average DPD / Creatinine (nmol / nmol) Standard Deviation Significant difference (*) One SHAM Vitamin K + D Supplement 210.3 19.1 b 2 OVX Vitamin K + D Supplement 281.6 27.9 CD 3 OVX 339.6 25.4 a 4 OVX n-3 PUFA 265.8 17.5 d 5 OVX Genistein 220.5 23.5 b 6 OVX Vitamin K + D Supplements n-3 PUFA Genistein 214.3 33.3 b (*) Different letters indicate significant differences between groups (p <0.05).

From the experimental data presented above, surprisingly, the ingestion of zenistein, n-3 PUFA and large amounts of vitamins K and D completely prevents the loss of bone mineral density caused by estrogen deficiency.

Claims (25)

(a) one or more isoflavones, one isoflavone glycoside or mixtures thereof; (b) one or more polyunsaturated fatty acids; (c) optionally a vitamin selected from the group consisting of vitamin D, derivatives of vitamin D, vitamin K, derivatives of vitamin K and mixtures thereof; And (d) optionally, auxiliaries and excipients in the range of 0.1 to 20% by weight, based on the required amount, preferably the total weight of the composition Comprising, comprising a composition useful for preventing osteoporosis. The method of claim 1, A composition selected from the group consisting of component (a) is a diedze, a diedze glycoside, a glycytein, a glycytein glycoside, genistein and a genistein glycoside. The method according to claim 1 or 2, Composition (a) is Genistein, Genistin or mixtures thereof. The method according to any one of claims 1 to 3, The component (b) is a polyunsaturated fatty acid of 16 to 24 carbon atoms comprising a plurality of unsaturated carbon-carbon double bonds. The method of claim 4, wherein The component (b) is a polyunsaturated fatty acid having 18 to 22 carbon atoms, preferably 18, 20 or 22 carbon atoms. The method of claim 4, wherein The polyunsaturated fatty acid is n-3 polyunsaturated fatty acid. The method according to any one of claims 1 to 6, Component (b) is selected from the group consisting of linoleic acid, linolenic acid, icosatetranic acid, icosapentaenoic acid and docosahexaenoic acid. The method according to any one of claims 1 to 7, Component (b) is (cis, cis, cis-) 9,12,15-octadecatenoic acid, (cis, cis, cis-) 6,9,12-octadecationic acid, (cis-) 5 , 8,11,14-icosaptranic acid, (cis-) 5,8,11,14,17-icosapentaenoic acid and (cis-) 4,7,10,13,16,19-docosa The composition selected from the group consisting of hexaenoic acid. The method according to any one of claims 1 to 8, A composition wherein the polyunsaturated fatty acid is at a concentration having a daily consumption in the range of 50 to 8000 mg, preferably 500 to 2000 mg. The method according to any one of claims 1 to 9, A composition comprising from about 5 to 200 mg, preferably from about 20 to 50 mg of component (b) for 1 mg of component (a). The method according to any one of claims 1 to 10, The composition (c) is selected from the group consisting of vitamin D ₃ , vitamin K ₁ and mixtures thereof. The method according to any one of claims 1 to 11, And vitamin D and vitamin D ₃ concentration of 5 to 1 day of vitamin D and vitamin D ₃ having the amount of consumption of each 50㎍ range, vitamin K, and each of the concentration of vitamin K ₁ 0.050 to 10mg, preferably 0.1 to 1.0mg Compositions of vitamin K and vitamin K ₁ having a daily consumption in the range. The method according to any one of claims 1 to 12, Wherein the line segment (d) is selected from the group consisting of starch, starch derivatives, cellulose, cellulose derivatives, polyols and mixtures thereof. The method of claim 13, Wherein the cellulose derivative is hydroxypropylmethylcellulose [HPMC], methylcellulose [MC] or mixtures thereof. The method according to any one of claims 1 to 14, A composition provided in a form selected from the group consisting of a paste filled with concentrates, granules, tablets, hard or soft gelatin capsules and foaming agents. The method of claim 15, A composition in the form of a concentrate that is a simple powder mixture of the components. The method according to claim 15 or 16, A composition in the form of a powder or granules obtainable by fluid bed granulation, high shear granulation, extrusion molding, spray drying or wet granulation. Claim 1, for the preparation of a composition or preparation from the group consisting of dietary compositions or herbal preparations, preferably reinforcing foods, reinforcing feeds, beverages, tablets, granules, hard or soft gelatin capsules, and foamable preparations. Use of a composition according to any one of claims to 17. A dietary composition comprising the composition according to any one of claims 1 to 17, preferably in the form of fortified food, fortified feed or beverage. The herbal preparation comprising the composition according to any one of claims 1 to 17, preferably in the form of a paste filled in tablets, granules, hard or soft gelatin capsules, or a foamable preparation. Use of a composition according to any one of claims 1 to 20 for preventing osteoporosis and stimulating bone formation in a mammal. A method of preventing osteoporosis and stimulating bone formation in a mammal comprising administering to the mammal an effective amount of the composition according to any one of claims 1 to 20. Use of a composition according to any one of claims 1 to 17 for the manufacture of a dietary composition or a herbal preparation for preventing osteoporosis and stimulating bone formation in a mammal. The method of claim 23, Use of a composition wherein the dietary composition or the herbal preparation is in the form selected from the group consisting of fortified foods, fortified feeds, beverages, tablets, granules, pastes filled in hard or soft gelatin capsules, and foaming agents. (a) genistein with a daily consumption in the range of 5-100 mg; (b) polyunsaturated fatty acids selected from the group consisting of linoleic acid, linolenic acid, icosatetranic acid, icosapentaenoic acid and docosahexaenoic acid; (c) a vitamin selected from the group consisting of vitamin D, vitamin D ₃ and mixtures thereof having a daily consumption in the range of 5-50 μg; (d) a vitamin selected from the group consisting of vitamin K, vitamin K ₁ and mixtures thereof, the concentration having a daily consumption ranging from 0.050 to 10 mg; And (e) optionally, adjuvants and excipients in the range of 0.1 to 20% by weight, based on the required amount, preferably the total weight of the composition A composition comprising a composition useful for preventing or treating osteoporosis and stimulating bone formation in a mammal.